Bacterial Cytochrome P450 Catalyzed Post-translational Macrocyclization of Ribosomal Peptides

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450-catalyzed RiPPs stand out as a unique but underexplored family. Herein, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence and co-conservation of precursors and P450s and interactions between them, successfully facilitating the identification of diverse P450-catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can catalyze the formation of Trp-Trp-Tyr (one C-C and two C-N bonds), Tyr-Trp (C-C bond), Trp-Trp (C-N bond), and His-His (ether bond) crosslinks, respectively, within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s to expand the chemical diversity of strained macrocyclic peptides and the range of biocatalytic tools available for peptide macrocyclization. P450 enzymes are highly effective catalysts, yet little research has been conducted on their ability to modify ribosomal peptides. Now uncharted bacterial cytochrome P450s responsible for the macrocyclization of ribosomal peptides have been discovered by sequence- and 3D-structure-based genome mining. These P450s exhibit diverse catalytic capabilities, catalyzing aromatic crosslinking by forming C-C, C-N, and C-O bonds.**+image